1. Field of the Invention
In general, the invention relates to the utilization in the field of human and veterinary medicine of a novel, sterile, essentially pure preparation of albumin. More specifically, the invention involves the utilization of albumin which has been disinfected by means of pretreatment with iodine to form a sterile albumin-iodine complex; the complex subsequently being subjected to an iodine recapture process thereby yielding a pure, sterile albumin product with enhanced binding properties.
2. Description of Related Art
Normal Human Serum Albumin is a sterile solution of serum albumin often prepared from pooled blood, plasma, serum or placentas obtained form healthy human donors. Each unit of plasma used in the preparation of albumin human must be nonreactive for hepatitis B surface antigen (HB-sAg), and the product is generally pasteurized at 60.degree. C. for 10 hours. Such albumin solutions contain no clinically important isoagglutinins or other antibodies and may be administered without regard to the recipient's blood group or Rh factor; none of the recognized components of the clotting mechanism of normal blood or plasma is present in albumin human solutions. AHFS Drug Information, 914, (1995)
Serum albumin is an important factor in the regulation of plasma volume and tissue fluid balance through its contribution to the colloid oncotic pressure of plasma. Albumin normally constitutes 50-60% of plasma proteins and, because of its relatively low molecular weight (66,300-69,999), exerts 80-85% of the colloidal oncotic pressure of the blood. Ibid.
The major clinical use of albumin is as a plasma volume expander and there are 50 years of experimental evidence to document its effectiveness. It is most widely used for its oncotic properties in the resuscitation of patients with an acutely diminished intravascular volume. It has beneficial effects on viscosity and is used for pump-priming in cardiopulmonary bypass surgery and hemodialysis. Although correction of an underlying disease process is necessary to correct defects in albumin production, exogenous albumin has been administered as an acute means of correcting hypoproteinemic states.
Albumin's properties as a transport protein have been used advantageously in binding bilirubin during therapy of hemolytic disease of the newborn. The Pharmacologic Approach to the Critically Ill Patient, 227 (1988) Albumin binds and functions as a carrier of intermediate metabolites (including bilirubin), trace metals, some drugs, dyes, fatty acids, hormones, and enzymes, thus affecting the transport, inactivation, and/or exchange of tissue products. AHFS Drug Information, 914 (1995) The importance of the inherent ability of albumin to bind various endogenous and exogenous substances accelerates as a foci for research dollars in a growing market.
Although albumin can bind a wide variety of substances in proportion to their lipophilicity, the protein has a particularly high affinity for weakly acidic substances, which at physiologic pH are present predominantly in the anionic form. Electrostatic forces are, therefore, likely to be important in binding, with additional contributions from hydrogen bonding and hydrophobic interactions. Binding is usually reversible, with association and dissociation time-constants typically being in the millisecond range. Mass action kinetics apply, predicting saturability at high ligand concentrations and competition among structurally related substances for common sites of interaction. Principles of Pharmacology, Basic Concepts & Clinical Applications, 49 (1995) There are three distinct classes of binding sites on the albumin molecule. Competitive displacement studies have been performed with warfarin, diazepam, and digitoxin as specific markers of each of these binding domains. Information so obtained has provided a useful framework within which to understand and predict drug-drug interactions attributable to alterations in binding to plasma albumin. Ibid
A well known example of the applicability of albumin binding sites to the detoxification of a neurological toxin involves the development of neonatal kernicterus after the administration of certain antibacterial sulfonamides. Early in the neonatal period, the breakdown of fetal hemoglobin releases large amounts of bilirubin that cannot be efficiently metabolized by conjugation to a more water-soluble metabolite due to the immature state of the hepatic enzyme, glucuronyl transferase in newborns. Bilirubin is extensively bound to albumin and this interaction (involving "warfarin-type" binding sites) is critical in preventing excessive increases in plasma concentrations of free bilirubin that, given the incomplete formation of the neonatal blood-brain barrier, can result in severe neurologic damage.
With regard to the present invention, it follows logically then, that if we can increase the number of available binding sites by administering a sterile, pure albumin which by nature of its production method, has a relative increase in the number of available binding sites in proportion to the number sites available on untreated albumin, we can effectively increase the margin of safety for patients against the possibility of bilirubin induced brain damage which results from displacement of toxic products from saturated binding sites on untreated albumin proteins.
The literature supports just such a proposition by reporting that, "In the treatment of hyperbilirubinema and erythroblastosis fetalis, albumin human is used as an adjunct in exchange transfusions; its bilirubin-binding activity reportedly reduces the number of transfusion required by increasing the amount of bilirubin removed with each transfuision ASHS Drug Information 914 (1995) This suggested use for the present invention of a purified albumin is intended to be representative only and is not meant to limit future or other uses of the product. Indeed, the use of albumin human as a transport protein to bind other toxic agents remains investigational and in light of the import of albumin binding and known albumin-drug and drug-drug interactions, the range of applications seems broad.
It is particularly in light of a demonstrable saturability of albumin binding sites that the import of one of the unique properties of my invention is revealed. Because albumin has the ability to bind any number of variable endogenous and exogenous entities, some of the aforementioned binding sites on untreated albumin can become saturated with superfluous agents added during the usual fractionation of plasma to produce albumin resulting in a concomitant decrease in the relative number of available binding sites on the protein molecule. A more efficacious application for this scarce resource can be realized with the prudent utilization of a sterile, purified albumin in which effectively all of the potential binding sites are available for use, thereby facilitating the ability of albumin to act as a carrier agent, either for the delivery of some product or for the removal of some toxic product according to the needs of the time.
The albumin product embodying this invention must be pure and sterile in order to maximize the number of available binding sites on the protein molecule itself. Prior inventors have taken numerous directions in pursuing various products with analogous desirable characteristics.
U.S. Pat. No. 5,346,992 describes a process for isolating albumin from supernatant IV, and in particular IV-4, or from COHN's fraction V or from plasma supernatant or fraction of analogous composition derived from an alcoholic or non-alcoholic anion exchange column with binding of albumin to the column, and then elution, and one step on a hydrophilic anion exchange column. Albumin thus obtained having a purity &gt;99% by cellulose acetate electrophoresis, being free from impurities detected by crossed immunoelectrophoresis and from polymers. Other similar ventures are described in that publication which have yielded purities of &gt;96%, 96.9%-98.9%, and &gt; or =99% and are incorporated herein by reference.
Previous albumin preparations suffered the problem of containing high amounts of aluminum as a contaminant along with it's associated toxicities, particularly in patients with impaired renal function who may be predisposed to suffering the detrimental effects of aluminum accumulation. U.S. Pat. No. 5,250,663 discloses a composition which comprises a solution of human serum albumin essentially free of chemicals used in processing. The preparation is also essentially free of metals such as aluminum. The composition is 100% pure by cellulose acetate electrophoresis and is essentially monomeric when tested by high pressure liquid chromatography. The preparation had a substantially longer shelf life and activity factor than other currently available products at that time. Novel applications of process methodology are taught in the preparation of this composition and a novel preparation result from essentially non hemoglobin containing albumin sources such as Source Plasma (Human) are discussed. Techniques and references are taught therein and are incorporated here by reference.
Once albumin has been separated from other various blood constituents, including other proteins, depending upon the nature of the origin of the albumin, there frequently remains other contaminants which need to be removed before the albumin is pure enough to meets the needs of the present invention. U.S. Pat. No. 5,250,662 provides a process for separating albumin from an impure protein fraction containing albumin. Contaminants, in an aqueous solution of the impure protein fraction containing albumin, are precipitated from the solution at a pH of from about 4.5 to about 4.7. Additional contaminants that remain soluble are bound to an anion-exchange resin. After the precipitated and anion-exchange-bound contaminants are removed from the albumin-containing solution, the pH of the solution is adjusted to from about 4.7 to about 6.1, and additional contaminants are precipitated. Further contaminants are then bound to an anion-exchange resin, and these precipitated and anion-exchange-bound contaminants are removed from the albumin-containing solution.
U.S. Pat. No. 4,197,238 discusses a method for purifying albumin, especially from human placenta, by removing blood group substance and hypotensive substances with polyethylene glycol to precipitate contaminant proteins containing blood group antigens and recovering albumin from the supernatant fluid. This patent discusses various methods of purifying albumin for infusion including many of the various shortcomings of these methods. The references from this patent are incorporated herein by reference.
An early patent, U.S. Pat. No. 4,169,829 includes a stepwise explanation of purification of albumin from frozen placenta, placental blood or other hemolyzed blood, by removing in a stepwise fashion, hemoglobin, enzymes and blood group substances. The value of this process is that when practiced according to the present invention, the albumin is well received by the receiving organism and does not cause antigenic and other reactions which may occur with prior art albumin preparations prepared from such raw materials.
The above methods of preparing albumin tend to yield product with problems or shortcomings in at least three areas. First, in all cases the albumin must still be "pasteurized" to insure a non-infectious product. Pasteurization is the process of subjecting the albumin obtained by whatever means to a heat treatment process wherein the product is sterilized by filtration and distribution aseptically into containers which are sealed to exclude micro-organisms and maintained at 59.5 to 60.5.degree. C. for 10 hours. Not only does this process kill many pathogens, but it also results in precipitation of a portion of the contaminating proteins which adversely affects the clarity of the final albumin solutions. However, to protect the albumin itself from precipitation additional substances such as caprylic acid are usually added. These additives bind to the albumin and stabilize it. Unfortunately, the final product then has at least some of its binding sites filled by the caprylate.
A second problem is that many of the above purification processes result in a lower overall yield of albumin due to the additional ethanol precipitation or the heating steps employed to ensure high purity. Furthermore, heating of the albumin results in an increase in the albumin polymer content however, high monomer contents are the desirable state of the art albumin product, since it has been suggested that albumin polymers are cleared more rapidly from the circulation than are albumin monomers, effectively resulting in the reduction of the concentration of infused albumin. It has also been suggested that the polymer form of albumin may produce an undesirable immunological response. See, U.S. Pat. No. 5,250,662.
Finally, overall albumin binding capacity is often compromised by the presence of endotoxin, a lipoidal material produced by bacteria. One of the natural functions of albumin is probably to bind such materials, but where do they come from during albumin fractionation under controlled laboratory conditions? The number of bacteria present in circulating plasma should be zero; however, when plasma is donated it becomes bacterially contaminated because it is impossible to maintain total sterility. If albumin is reclaimed from placenta or cord blood, the possibility of bacterial contamination is even higher. Although the blood or plasma is generally refrigerated during processing, considerable bacterial growth still occurs. Although these bacteria are ultimately killed by pasteurization or other steps, they still release considerable endotoxin which then binds to the albumin.
The current inventor has explored the use of iodine complexes in the treatment of blood and blood complexes in considerable detail. For example, the inventor's U.S. Pat. No. 5,370,869 discloses a method of disinfecting platelet-bearing liquid by contacting the liquid to be cleansed with solid povidone-iodine to expose the platelet-bearing liquid to iodine and thus kill pathogenic organisms therein, and thereafter removing the liquid from contact with the solid povidone-iodine.
Additional aspects of the inventor's work are reported by Highsmith et al., Blood, 86(2): 791-96 (1995). This study explored the use of liquid iodine for inactivation of several lipid and nonlipid enveloped viruses in an antithrombin III (AT-III) concentrate. Iodine at levels of about 0.01% to about 0.02% caused between 43% and 94% loss of AT-III activity, as well as degradation of AT-III as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.
If the use of albumin in the medical and veterinary arts is truly to be maximized, simply producing a pure albumin does not address all of the concerns of those who would employ it. That is, not only is it desirable for the albumin to be pure, but it must also be sterile. We live in a world of increasing risk with regard to transmitted diseases and their growing resistance to antimicrobial therapy. Gone are the days when the administration of a single injection of a drug such as penicillin could cure even the most woeful diseases. Today, we are faced with challenges and risks that carry the gravest consequences. Unfortunately, even as of this writing, infection with the AIDS virus leaves only the slimmest hope for normal longevity. The author in U.S. Pat. No. 5,204,324 has addressed a technique for inactivating at least one of the more prevalent pathogenic viruses found in animal fluids and tissues.
Clearly, a method of producing not only a pure, but also a sterile albumin which would allow maximal use of available binding sites would represent a valuable asset in various albumin-type therapeutic interventions.